Monitoring the battleground: exploring antimicrobial resistance and virulence factors in wound bacterial isolates

Antibiotic resistance poses a grave global public health threat, exacerbated by widespread and often inappropriate antibiotic usage. Vigilant surveillance of antibiotic utilization and emergence of antimicrobial resistance (AMR) is essential. Of particular concern in the era of AMR is the persistent issue of chronic wound infections. To address this, we conducted a comprehensive evaluation of wound isolates from chronic wounds at Jaramogi Oginga Odinga Teaching and Referral Hospital (JOOTRH) in Kenya, to identify relevant bacteria and assess their drug resistance patterns.Wound samples were collected and processed using standard microbiological methods. Bacterial isolates were identified and assessed for their susceptibility to a panel of antibiotics using the Kirby-Bauer disk diffusion method. A total of 103 bacterial isolates were obtained from the wound samples, with a higher prevalence in male patients (59%). Staphylococcus aureus (20.7 %) emerged as the most predominant pathogen, followed by Klebsiella spp. (14.8 %), Pseudomonas aeruginosa spp. (14.8 %) and Escherichia coli (4.4 %) in wound samples. High levels of antibiotic resistance were observed among the isolates, with the highest resistance rates reported for cotrimoxazole (48.1 %), clindamycin (25.9 %) and erythromycin (25.9 %). Furthermore, among the isolates, 75 % produced haemolysin and protease, while 50 % produced lipase and phospholipase, factors that enhance virulence and survival. The findings of this study highlight the alarmingly high prevalence of antibiotic resistance among bacterial pathogens isolated from chronic wounds in Kenya. This poses a major challenge to the effective management of chronic wound infections. There is an urgent need to implement effective antimicrobial stewardship programs and develop new antibiotics to combat the growing threat of antibiotic resistance.

microbial assemblages which live on it [7,8].Bacterial infections, whether through contact with contaminated water, trauma, accidents, surgical operations or burns can result due to any breach in the skin surface [7].A diverse array of microorganisms ranging from bacteria to fungi and parasites and viruses can infect wounds [9,10].These organisms can lead to the formation of wound biofilms, which further complicates wound healing as biofilms have increased antibiotic resistance [10].
Unfortunately, control of wound infections has become more challenging due to widespread bacterial antibiotic resistance and to a greater occurrence of infections caused by methicillin-resistant S. aureus (MRSA), polymicrobial flora [11].Although the development of antimicrobials in the 20th and 21st centuries transformed the treatment of human infections, the rapidly increasing antimicrobial resistance poses a threat to mitigating infections.Managing the increasing range of infections caused by microorganisms remains a dynamic risk due to antimicrobial resistance [12].Hence the management of patient demands and increasing costs is leading to persistent infections and increasing deaths due to since the reduced effectiveness of antibacterial drugs [12].Antimicrobial resistance is increasing in incidence due to the burden of using and misapplying antibiotics [13].There has been an emerging desire for the development of new antibiotics that are more effective in the management of resistant bacteria [13], due to common bacteria developing resistance to recently discovered antimicrobials [13,14].The absence of new antibiotics to replace those that are no longer effective is given more urgency with the desire to guard the effectiveness of current medications, and advance and enact suitable methods to curb the rise and spread of antimicrobial resistance [15].
In developing countries, antimicrobial resistance poses a great threat to community health care, leading researchers to determine the resistance profile of antibiotics against bacteria.Nevertheless, the conclusions continue to produce different opinions on the effectiveness of some drug.For instance, studies in Libya [16,17], as in Sudan [18], revealed sensitivity of isolates obtained from wounds at rates of 54 % with amikacin and 59 % with ciprofloxacin but resistance at rates of 81 % with vancomycin, 75 % with amoxicillin, 92 % with streptomycin, 45 % with tetracycline, 26 % with methicillin, 65 % with amoxicillin and 46 % with erythromycin.On the other hand, bacterial isolates obtained from wounds were highly resistant (96 %) to ciprofloxacin in Nigeria [19], which contradicts other findings.
The growing complication of antimicrobial resistance remains a disturbing problem worldwide, motivating boundless research and novel approaches to research on bacterial susceptibility and resistance to antibiotics is a priority subject, sometimes producing contradictory results.For instance, in wound management, Pokhrel et al. [19], and Gupta et al., [20] recognized that S. aureus isolates were therapeutically responsive to amoxicillin clavulanate, meropenem, clindamycin, ceftriaxone, piperacillin-tazobactam, ciprofloxacin, vancomycin, levofloxacin, linezolid, teicoplanin, imipenem, meropenem, amikacin and Levofloxacin but resistant to ampicillin, amoxicillin clavulanate, cotrimoxazole, doxycycline and cephalosporins.Other studies revealed high resistance to cephalosporins, amoxicillin clavulanate and imipenem [20,21].Hence, the present study evaluated bacterial isolates from patients with wound infections and drug susceptibility patterns were examined, with the goal of deciphering antibacterial resistance, from which some resistant isolates were identified based on commonly used antibiotics.

mETHoDS
We examined all samples received at the Medical Microbiology Laboratory Department of Jaramogi Oginga Odinga Teaching and Referral Hospital (JOOTRH), Kisumu county, Kenya, for the period of May to August 2022.Samples (wound pus/swab) were obtained from the patients who were seeking medicare at JOOTRH both in outpatient and in inpatient departments; patients referred to the hospital were not included in this study.
monitoring the presence of relevant virulence factors of P. aeruginosa spp.

Detection of haemolysin
Haemolysin production by the P. aeruginosa spp.isolates was detected following the protocols given by Benson et al. [26].Betahaemolytic activity was tested for on base agar (Himedia) supplemented with 7 % sheep erythrocytes for 18-24 h.Pure isolates were cultured on TSA, before streaking on BA and further incubated for 24 h at 37 °C.Zones of haemolysis around the colonies after 24 h indicated the ability of these bacteria to haemolyse red blood cells (RBCs) [27].

Detection of protease
To detect protease production by the P. aeruginosa spp., skim milk agar was used and a previously described protocol [26].Briefly, two solutions (A and B) were made and used in this study.Solution A was prepared by adding 10 g skimmed milk to 90 ml distilled water, the volume was then completed to 100 ml, gently heated at 50 °C, then autoclaved and cooled to 50-55 °C.
Solution B was also prepared by adding 2 g of agar powder to 100 ml distilled water, mixed thoroughly, then autoclaved and cooled to 50-55 °C.Aseptically, 100 ml of solution A was mixed with 100 ml of solution B. The mixture was then poured into sterile Petri dishes, and then stored at 4 °C until use.This medium was used to detect the ability of the bacteria to produce protease [28].The appearance of a cleared hydrolysis zone indicated a positive test [29].

Detection of lipase
Lipase production ability by P. aeruginosa isolates were determined by methods outlined by Elliot et al. [29].Briefly, a single colony of an overnight growth was cultured on Rhan medium, and then incubated for 1-5 days at 37 °C.The appearance of a turbid zone around colonies on the fourth day indicated a positive result [26].

Detection of lecithinase (phospholipase)
To detect lecithinase, we followed a standard procedure [30].One pure colony was cultured on the medium used for phospholipase activity assay followed by incubation for 1-3 days at 37 °C using established procedures [31].The appearance of a white to brown elongated precipitated zone around colonies on the second day was considered a positive result [32].

Validity and reliability
All experiments were conducted in triplicates that were independent of each other to validate reproducibility.

Data analysis
Statistical analysis was performed using SPSS version 20.Data on socio-demographics were summarized by frequencies and percentages.All values of diameter zones of inhibition are reported as mean±se.
Among 37 (27.4 %) Gram-positive isolates, 28 (20.7 %) S. aureus resistance patterns revealed that the most effective antibiotics were cotrimoxazole [13 (48.1 %)] followed by clindamycin [7 (25.9 %)] and erythromycin [7 (25.9 %)] while lower resistance to cotrimoxazole [3 (37.5] %) was observed with Staphylococcus spp.Among 36 (26.7 %) Gram-negative isolates, 20 (14.8 %) Klebsiella spp.resistance patterns revealed that the most effective antibiotic was tetracycline [11 (61.1 %)] followed by gentamicin at [9 (50 %)] while for P. aeruginosa four isolates showed multi-drug resistance (MDR) to commonly used antibiotics, while for E. coli the isolates were sensitive to almost all of the common antibiotic used in the facility for their management, as shown in Table 3 and Supplementary data 2. A-C.Of 10 P. aeruginosa isolates, four strains were MDR, with one strain was resistant to three classes of antibiotics (12583), two strains were resistant to five classes of antibiotics (13642 and 14421) and one strain was resistant to six classes of antibiotics (11985).
This study investigated the production of various virulence enzymes such as protease, phospholipase, lipase and haemolysin on the four P. aeruginosa isolates which showed MRD and were found to be resistant to common antibiotics used for its management within the study area.Of the 10 isolates four showed resistance to different antimicrobial classes, and one of which, isolate 14421, was able to produce all types of the virulence enzymes.It was also confirmed that isolate 12583 was able to produce all enzymes except phospholipase while isolate 11985 was also capable of producing all except lipase.Lastly, isolate 13642 was not able to produce any of the virulence enzymes, as shown in Table 4.

DISCuSSIon
Antibiotic resistance is a global health concern, necessitating ongoing monitoring.This study is a response to the pressing need for continuous surveillance of antibiotic resistance trends, especially in the context of chronic wound infections, which remain a public health concern.The study focused on patients with chronic wounds seeking medical care at Jaramogi Oginga Odinga Teaching and Referral Hospital (JOOTRH) in Kenya.By collecting and analyzing wound samples, the research aimed to uncover the prevalence and patterns of antibiotic resistance among bacterial pathogens in this specific population.The findings were both particularly interesting and alarming.Out of the 135 samples collected from patients with wound infections, 73 (54.1%) exhibited positive bacterial growth, indicating a substantial burden of microbial infections.The isolates included a range of bacteria, with Staphylococcus aureus, Klebsiella spp., Pseudomonas aeruginosa and Escherichia coli being the most prevalent.These pathogens are known to be responsible for various wound infections and can pose significant challenges in treatment.In similar studies conducted previously [22] on antibiotic susceptibility patterns of bacterial isolates causing wound infection among patients visiting two hospitals in India [24], positive growth rates were 44.9-50%.Separate studies [25,33] in a tertiary hospital in South Africa showed more than 90 % growth emphasizing the eminent concern.The positivity rate was higher in male patients for both Gram-positive and Gram-negative isolates, 23 (29.1 %) and 26 (32.9 %) respectively.A similar study [34] agreed with our findings.The relatively higher percentage of male patients might be due to active involvement of males in outdoor activities, including agricultural work, resulting in high possibility of infection and prevalence of high rates of accidents [26].Our findings underscore the concern of a significantly elevated positivity growth rate, potentially affecting the wound healing process, particularly since many samples contained multiple isolates.Conversely, despite the precautions taken in such studies, it remains uncertain whether some cases of culture-negative results can be ascribed to non-infectious bacteria, errors in sample collection, transportation challenges, delays in processing, or the prior use of antibiotics by patients.
Further, the data herein revealed intriguing insights into the prevalence of wound infections and antibiotic resistance patterns in different age groups.We found the highest rate of positivity in the age group >45 years, contrary to previous studies that reported the highest growth rate in the 21-30 years age group [26,33].This disparity might be attributed to the elevated presence of older individuals in our study, potentially resulting from increased activity and accidents leading to hospital admissions in this age category.From the total bacterial isolates, 37 (27.6 %) were Gram-positive and 36 (26.9 %) were Gram-negative bacteria.This result did not match with previous studies [21][22][23][24][25][26][29][30][31][32][33][34][35] which showed Gram-negative bacteria being predominant since their peptidoglycan layer is much thinner than that of Gram-positive bacilli, and they are harder to eradicate because when their cell wall is disturbed they release endotoxins that can worsen patient symptoms [24].Our observation of Gram-negative bacteria beinmg the most common in wound infections differs from other studies in Nigeria reporting Gram-positive bacteria to be predominant [29].Among the Gram-negative pathogens, Klebsiella spp. was the most predominant [20(14.8%)]; the pathogenicity of K. pneumoniae is mediated by several virulence factors that allow it to evade host innate immune responses.These factors include the capsule, lipopolysaccharide, adhesions, iron acquisition systems, resistance to serum and biofilm formation [34].Among Gram-positive taxa, S. aureus was the predominant isolate [28 (20.7 %)] and the most predominant among the total.S. aureus pathogenicity is mediated by bacterial components and secreted virulence factors such as surface-associated adhesions, capsular polysaccharide (CP) and exotoxins [29].This finding did not match a similar study conducted [32], where P. aeruginosa was the most predominant bacterium (29.9 %) among the total isolates, which may be due to the permeability barrier afforded by its Gram-negative outer membrane, while S. aureus was predominant (27.5 %) among Gram-positive isolates.In a previous study [32], Gram-negative bacilli constituted 66 % of all the pathogens with P. aeruginosa as the most frequent (19 %).A higher prevalence of S. aureus among Gram-positive bacteria and the most predominant isolate among the total seen in this study has also been reported by other researchers [10,36],.These results shed light on the diversity of wound infections and the potential regional variations in bacterial prevalence, emphasizing the need for tailored treatment strategies.Moreover, they underscore the persistent challenge of antibiotic resistance and the importance of responsible antibiotic use to combat these infections effectively.
In the susceptibility patterns towards Gram-positive isolates, S. aureus isolates showed an alarming resistance to cotrimoxazole (48.1 %), a commonly used antimicrobial drug in wound management.Studies have shown that S. aureus can become drugresistant either via genetic mutations on DNA gyrase or through reduced expression of outer membrane proteins reducing drug accumulation [53].The findings align with earlier research, corroborating resistance rates of 47% and 49% to cotrimoxazole [37][38][39] .Cotrimoxazole has traditionally served as a first-line treatment for wound infections and has been extensively prescribed for gastrointestinal, respiratory, urinary, and skin infections.Its mechanism of action involves targeting a DNA gyrase subunit, crucial for bacterial DNA synthesis [39].Furthermore, it is frequently administered to immunocompromised patients, such as those with HIV, which has a prevalence of 16.3% in the study area [38].The widespread usage of cotrimoxazole, its affordability, and its accessibility without a prescription likely contributed to the emergence of resistance, as observed in our study.This underscores the urgent need for prudent antibiotic use to combat the growing challenge of antimicrobial resistance.
A notable occurrence of resistance was observed among S. aureus isolates, with a resistance rate of 25.9% against erythromycin.
Erythromycin is a broad-spectrum, bacteriostatic antibiotic effective against various Gram-positive bacteria.Its mechanism of action involves irreversible binding to a receptor site on the 50S subunit of the bacterial ribosome, thereby inhibiting peptidyl transferase and preventing the transfer of amino acids to growing peptide chains, ultimately hindering protein synthesis.Until late 2018, erythromycin was the primary treatment for conditions like typhoid and Salmonella infections [32,35,40].However, because of resistance and safety issues, it is no longer the first-line treatment in enteritis.In low-income countries, it is still widely used, as it is not expensive and is readily available [27].Erythromycin has been recommended by the World Health Organization (WHO) for the treatment of wound infections in both children and pregnant women [41].The high resistance observed may be explained by its frequent usage for the treatment of severe coughs and other infectious diseases.Also, erythromycin resistance was found to be due predominantly to the presence of an Erm (B) methylase.Other reports have indicated that S. aureus isolates are resistant to erythromycin [37].A similar incidence was observed for other pathogens causing diarrhoea in a study conducted in the study area [42,43].
Additionally, Klebsiella spp.demonstrated a notably high resistance rate of 61.1% to ampicillin, exceeding that of gentamicin at 50%.Ampicillin targets penicillin-binding proteins anchored in the bacterial cell membrane, essential for cell wall cross-linking.Klebsiella spp.isolates in previous wound infections have been recognized for their resistance to ampicillin, gentamicin, and imipenem [44].There has been reported to be an emergence of P. aeruginosa isolates resistant to ampicillin (33.3 %), gentamicin, amikacin and ceftriaxone (all at 20 %), with 10 isolates resistant to this antimicrobial, which has been primarily associated with the presence of Inc C conjugative plasmids [41], hence leading to four isolates being found to be MDR.Among the MDR isolates, two showed resistance to three antimicrobial classes, one resistance to four antimicrobial classes, two resistance to five classes and finally one resistance to six antimicrobial classes.Studies on MRSA have shown their wide variation.Naik and Deshpande [45,46] showed an 8.0 % prevalence of MRSA, which agrees with our study.All E. coli isolates were sensitive towards all the antibiotics used except lower resistance to imipenem, thus confirming the higher efficiency of these agents against E. coli isolates at Kisumu County.
Surprisingly, of four isolates of P. aeuruginosa, three (75 %) (12583, 14421 and 11985) were able to produce haemolysin, a finding that did n't conform with a previous study [28] which showed 100 % isolates producing the enzyme.As stated previous, purified haemolysin can cause fluid accumulation [32]; in contrast to the watery fluid produced in response to Cholera Toxic (CT), the accumulated fluid produced in response to haemolysin is invariably bloody with mucus [32].Also, of the four isolates, two (50 %) (12 583 and 14421) had the ability to produce lipase.Our findings also concur with other studies, which showed that 50 % f isolates obtained in the study had the ability to produce lipase [16].Lipase enzymes catalyse the hydrolysis of the ester bonds of triacylglycerols and may have a critical role in P. aeruginosa pathogenicity or nutrition acquisition.The production of excess lipase allows bacteria to penetrate fatty tissue with the consequent formation of abscesses [45,47].The production of these enzymes by the isolates may reflect the presence of genetic organization of a discrete genetic element, which encodes three genes responsible for producing proteases, lipases and phospholipase.This organization could be a part of a pathogenic island, encoding a product capable of damaging host cells and being involved in nutrient acquisition [48].These findings underscore the urgent need for responsible antibiotic use and the development of more effective antibiotics to address chronic wound infections and the growing antibiotic resistance threat.

ConCLuSIon AnD RECommEnDATIonS
AMR isn't merely an impending crisis; it stands as an immediate global emergency, imperiling our capacity to effectively combat infections, conduct surgical procedures, and manage diseases that were once easily treatable.It represents a silent pandemic, stemming from the excessive and inappropriate use of antibiotics, inadequate hygiene and sanitation practices, and a dearth of innovative antimicrobial solutions.The findings of this study underscore a deeply troubling trend in antibiotic resistance among both Gram-positive and Gram-negative bacteria, resulting in treatment failures.These resistant strains specifically generate various virulent enzymes, including haemolysin, lipase, protease, and phospholipase C, culminating to bacterial resistance.Furthermore, despite inhibitory measures, the study's results reveal the capacity of these antibiotic-resistant strains to create biofilms.As AMR escalates across a wide spectrum of microorganisms, there is an urgent, global imperative to enhance antimicrobial treatments and pioneer innovative strategies for developing new antimicrobials.This entails utilizing innovative tools such as gene-mining applications and high-throughput genetic sequencing, along with the integration of computational tools and artificial intelligence to identify novel antimicrobial agents.Moreover, the collection of clinical data on AMR assumes paramount importance for the monitoring of resistance trends and in the development of superior antimicrobial drugs.This coupled with the adoption of forward-thinking approaches like artificial intelligence will expedite the discovery of new drugs and prioritize the issue of mounting antibiotic resistance in bacteria, including within the realm of chronic wound management.The microbiology Society is a membership charity and not-for-profit publisher.
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Comments: Thank you for adding clarification to the biofilm methodology.The additions mean that others would be able to replicate the work.There are still two outstanding issues.1) The AMR data with respect to the description of strain 13642.Please amend this to remove ambiguity.The sentence should read "out of 10 P. aeruginosa isolates, 4 strains were MDR, with 1 strain resistant to 3 classes of antibiotics (12583), 2 strains resistant to 5 classes of antibiotics (13642 and 14421), and 1 strain resistant to 6 classes of antibiotics (11985)".Table 4 is also redundant-please remove it.
2) The representation of the biofilm data are also problematic.The new combined graphs look great, thank you for resolving this issue.However, the comparison to an unrelated PC strain is not methodologically sound ("The P. aeruginosa isolates showed that the biofilm formation inhibitory effects of the various concentrations (0.5, 0.25, 0.125, 0.0625 and 0.03125 mg ml−1) were significantly lower than that of the positive control, an indication that biofilm formation was inhibited at these concentrations").I can see from the methods section that you describe how Pa% antibiofilm activity is calculated ("Abf A (%) = (1-(ODTest sample -ODBlank)/ (OD Untreated sample -OD Blank) × 100"), and there is nothing wrong with that.The problems come in when calculating the statistical significance in relation to PC strain P. aeruginosa ATCC 12934.I understand the inclusion of this strain into your biofilm assays to ensure biofilm is formed, but the comparison of the antibiofilm activity against each strain should be compared to the untreated sample.Please recalculate these P values making the comparison between untreated samples vs treated samples, not treated samples vs control strain.The paragraph describing these results (lines 246-260) need to be rewritten as they are currently not as comprehensible as the rest of the manuscript.Comments: Thank you for attempting to address the most recent comments on this manuscript.However, there are still several issues that are not fully resolved, as detailed below; Point 1) Thank you for adding clarification to the statistical analysis section.

Response to reviewer verdicts
Point 2) The text in manuscript reads-"Then a colony was identified, picked and inoculated in 10 ml of LB broth and incubated at 37 ˚C 48 overnight while shaking at 100 r.p.m. for 18 h.By use of a parafilm the flat-bottomed polystyrene tissue culture microplate was sealed for purposes of preventing medium evaporation." Information is still missing from this method.There is a step missing in between taking a single colony and culturing it in 10 ml broth overnight, to then sealing the plate with parafilm.What volume of culture do you place in each well?And what is the culture density-do you normalise the cultures to a particular OD or CFU before putting it into the wells of the plate and sealing it with parafilm?If you don't normalise the starting inoculum, then how do you compare across plate replicates and in between strains?Point 3) I'm afraid I still don't understand the MDR data presented in figure 1. Can you clarify why strain 13642 gets grouped with strains that show resistance to 3, 4, and 5 classes of antibiotics.Surely, they should just be grouped into strains which are resistant to 5 classes of antibiotics as it is therefore a given that they show resistance to 2, 3, and 4 classes of antibiotics because they are resistant to 5 classes in total.So out of 10 P. aeruginosa isolates, 4 strains were MDR with 1 strain resistant to 3 classes of antibiotics (12583), 2 strains resistant to 5 classes of antibiotics (13642 and 14421), and 1 strain resistant to 6 classes of antibiotics (11985).(with no need for figure 1 to be presented).Point 4) Thank you for attempting to put together the biofilm data into one single figure instead of 3 separate ones.However, the figure looks like it has been cut and pasted from the original graphs rather that being replotted which makes the figure messy and the resolution and scale of each plot doesn't match.Before publication these will need to be replotted.Additionally, upon looking at this biofilm data to check the plots I have noticed something important that wasn't flagged in the peer review reports.The use of strain ATCC 12934 at the "positive control" and comparative strain in your anti-biofilm treatment graphs is inappropriate.This strain has been used as the benchmark to test the ability of 6 different antibiotics to disrupt/prevent biofilm formation by strains 12583, 14421, and 11985, but the true comparative assay should be each of those strains with no antibiotic added.This would give you the baseline biofilm formation by these strains and allow you to see how well each antibiotic disrupts the biofilm of each strain.For instance, your data make it look like strain 11985 is more sensitive to the antibiofilm activity of all antibiotics tested compared to the other two strains, but how do you know it isn't just a weaker biofilm producer in the first place?These antibiofilm experiments need to be completely redone to include each strain with no antibiotic added.If you are not able to redo these experiments, I would suggest removing all of the phenotypic characterisation of P. aeruginosa data completely as it is not clear why you have chosen these strains for characterisation out of all 135 bacteria you isolated from wounds to begin with.
What was interesting about the 10 P. aeruginosa strains that led you to do AMR, antibiofilm, and virulence phenotyping-this was a question raised by reviewer 1 in their original peer review (result point 4) but hasn't been made clearer in your revisions.

Date report received: 31 May 2023 Recommendation: Major Revision
Comments: This manuscript gives an overview of AMR persistence in patients with wounds in a Kenya hospital.The study appears to be well designed, but the manuscript needs to be improved.The Introduction section needs an in-depth revision to be up to the standards of the journal.For instance, lines 61-65 would benefit from being re-written and important review papers (like https:// pubmed.ncbi.nlm.nih.gov/ 35065702/) should be cited.Additionally, lines 67-70 need to be made clearer.The last two paragraphs are particularly confusing and one of the papers cited (19) cannot be accessed, which obfuscates the point the authors try to make.The Methods section is thorough and allows for good reproducibility, with good description of the protocols and controls used.Note that at the end of line 120 the word "well" is missing.The Results section is a bit confusing mainly due to the structure of the sentences and the use of percentages when it doesn't add extra information, as well as several mistakes (see below).I would also like the authors to clarify if any of the samples analysed had both gram positive and gram negative bacteria.1) In Table 1 the row for "Median Age" is not formatted correctly and in my opinion doesn't add any relevant information so it should be removed (or its relevance should be made apparent). 2) The text and Table 2 refer to 135 samples, but Table 1 to 134 samples, which has implications in the accuracy of all percentages calculated in the text and tables (currently they don't match, and this should be corrected).3) Paragraph from lines 202 to 206.It states that only 4 isolates are MDR but describes 6 isolates since isolate 13642 was included in three categories (resistant to 3, 4 and 5 antimicrobial classes).This mistake is then translated into Figure 1. 4) Table 4 is confusing as it has +/-but also p/n for positive/negative.Authors should choose only one nomenclature (usually it's +/-) and rewrite paragraph from lines 210 to 218 since it is confusing and particularly lines 213/214 don't make sense.5) Figures 2-4 should be converted into a single summary figure using colours to distinguish the 4 isolates, and in this way facilitate the comparison between isolates.The text that describes those figures is also very hard to understand, with sentences that don't make sense (for instance lines 229 to 231).Since the authors compare results of the biofilm formation inhibition assay with the antimicrobial sensitivities for the 4 P. aeruginosa MDR isolates, the specific resistances for these isolates should be presented in a table alongside the summary figure (including data now in Figures 2-4) as to facilitate interpretation.Lines 236/237 should be included in the discussion, not in results.The Discussion section is written in a very convoluted way, particularly the initial paragraphs.It doesn't help that the citations appear not to match what the authors wanted to cite (for instance, references 22, 25 and 26 refer to methods and not studies, or reference 29, that is referred in text as a study from Nigeria, is actually a book chapter about Vibrio).I suggest the authors re-write this section and go straight to the point (for instance, in the first paragraph the information could be summed as: "In our study, X% of the samples tested were positive, in line with the results from studies Z and W that showed z% and w% respectively").Furthermore, from line 348 authors focus on the production of different enzymes by the 4 MDR isolates, and compare these with other studies, but with such a small number of samples, conclusions such as "finding didn't conform with previous studies" cannot be made.Authors should refrain from using colloquial names for effects, and in my opinion the sentence on lines 365/366 should be removed.The Conclusion section includes good points, but a careful revision of the text should be done to allow better readability.

Please rate the quality of the presentation and structure of the manuscript Poor
To what extent are the conclusions supported by the data?Strongly support

Is there a potential financial or other conflict of interest between yourself and the author(s)? No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes Comments: The author's present a good manuscript detailing the use of antibiotics for bacterial infections, focussing on bacteria associated with wound discharge such as P. aeruginosa and S. aureus.This work will add to the literature to provide insight into the phenotypic characteristics of wound-associated bacteria and antibiotic use for chronic wound infections.Whilst this is a good start, I do feel there are some things that the author's could improve on in the manuscript which are described below.Methods: 1) Check for correct spellings throughout manuscript.For instance, a dash in Mac-conkey is not needed, as it should read MacConkey 2) On line 98, the author's describe the plates to be incubated between 24-48 hours growth, which is a huge difference.Could this be more defined as to which plates were incubated to what specific time point.3) Were any specific bacteria targeted during the screening?It is confusing when the methods jump straight to saying "Isolated S. aureus was screened".Could a table be used to describe the bacteria isolated and the number/percentages of these?4) Could the author's further define what statistical analyses were done i.e. was it parametric/non-parametric.I did notice that the statistical analysis sections also contradict each other -in the main methods section it says that SPSS/GraphPad was used, but in the statistical analysis section it says that Stata was used.Could the author's please make it clear which one was used/if different statistical analysis packages were used for different experiments?5) Line 111 -what size microtiter plate was used? 6) For antibiotics tested

Point 2 :Point 3 :Point 4 : 3 Editor
Question raised on turbidity standard is factored in as shown in point roman 1, then volume of culture put on each well was as indicated in roman 2. I. Bacteria was standardized to attain the MacFarland turbidity standard of 0.5 ×106 CFU/mL.Line 155-157.II.190 µls of LB broth with various dosages of the various antibiotics (0.5, 0.25, 0.125, 0.0625 and 0.03125 mg ml −1 ) were then inoculated with 10µls of bacterial cultures per well of the flat-bottomed polystyrene tissue culture microplate.Line 156-157 On this I mean the strain was able to show resistance on the different classes, after which I resolved it as per the updated shared document on line 226 to 232.And further replace the figure with Table 4 as in line 234 Since this graph deals with different isolate on different drugs bringing this into one graph is a big challenge but kindly have a look at the final copy on line 260-262 NB:I settled on P. aeruginosadue to its multidrug resistance towards different classes of antibiotics unlike the other isolates and out of the 10 isolates I was only interesting on the 4 isolates as shown in my findings line 262-232 VERSIon recommendation and comments https://doi.org/10.1099/acmi.0.000613.v3.1 © 2023 Rudkin J.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Justine Rudkin; University of Oxford, Nuffield Department of Population Health, UNITED KINGDOM, Oxford Date report received: 15 August 2023 Recommendation: Major Revision

Table 1 .
Sociodemographic characteristics of patients involved in the study for the period of May to August 2022

Table 3 .
Antibiotic resistance patterns of isolated pathogens

Table 4 .
Detection of some virulence factors of P. aeruginosa Naik G, Deshpande SR.A study on surgical site infections caused by Staphylococcus aureus with a special search for methicillinresistant isolate.J Clin and Diag Res 2011;5:502-508.46.Ammons MCB.Anti-biofilm strategies and the need for innovations in wound care.Recent Pat Antiinfect Drug Discov 2010;5:10-17.47.Abbass NBM.Effectiveness of Some Physical and Chemical Factors on the Morphological Changes of Vibrio cholerae Isolated from Environment.Ph.D. Thesis.University of Al Mustansyria, Iraq; 2006.
The biofilm data is emerge into a single figure as shown in line 263-270, but by but luck the Graph Pad Prism version 9.4 I used for analysis had no section of the clear colour option as requested hence I'm not able to adjust the colour more than as shown in line 263-270.We think sharing the same data on data form could be a repetition of same work therefore we opted for figure interpretation over the table as shown.I.All tables changed into figures are all turn back into tables as it was before and this can be seen throughout the manuscript under result.II."Growth rate"-this words have been restructured into the highest rate of sample positivity throughout the result discussion section.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.I have taken a detailed look at the changes you have made, which wasn't a straight forward task as the track changes document doesn't contain any changes beyond the abstract.However,I am afraid I am going to have to send this manuscript back to you as several points have not been sufficiently addressed, and a few of the tables have now been changed into figures which are poorly labelled.Key points from the original review that need to be addressed; 1) Please specify what statistical tests have been used through out.This question was asked by reviewer 1 but was not addressed.2)Point7raisedbyreviewer1hasnotbeenfully met-please specify the inoculum used to seed the microtiter plates in the biofilm assays.3)Point 3 raised by reviewer 2 has not been addressed-"Paragraph from lines 202 to 206.It states that only 4 isolates are MDR but describes 6 isolates since isolate 13642 was included in three categories (resistant to 3, 4 and 5 antimicrobial classes).This mistake is then translated into Figure1."(which is now figure5).How can the same isolated been included in the category of resistant to 2 classes, 3 classes, and 4 classes of antibiotics?4)Point 5 raised by reviewer 2 about merging the biofilm data into a single figure has not been actioned-"Figures2-4should be converted into a single summary figure using colours to distinguish the 4 isolates, and in this way facilitate the comparison between isolates.The text that describes those figures is also very hard to understand, with sentences that don't make sense (for instance lines 229 to 231).Since the authors compare results of the biofilm formation inhibition assay with the antimicrobial sensitivities for the 4 P. aeruginosa MDR isolates, the specific resistances for these isolates should be presented in a table alongside the summary figure (including data now in Figures2-4) as to facilitate interpretation".This was something I specifically drew attention to in my original decision letter and something which would greatly improve the presentation on the results.See these manuscripts for good examples of what it could look like https:// journals.asm.org/doi/pdf/ 10. 1128/ AAC.02885-15 , https://www.microbiologyresearch.org/docserver/ fulltext/ jmm/ 66/ 3/ 377_ jmm000446.pdf?expires= 1689773745& id= id& accname= sgid025019& checksum= 5478 2BB9 4A5B 25C9 34FD F7C6 DC6C7E70 Additionally, I am not sure why tables 1 and 2 have now become figures?They don't convey the information as well as the tables did, and the text in the legend and the key do not match-"Gram +ve and Gram -ve cases are represented in light-blue and dark-blue, respectively while cases where neither bacteria strains were observed are shown in light grey".Please revert back to the original tables which were much more informative and easier to extract information from.Lastly, there is talk throughout the manuscript of growth rates to mean incidences of positive growth of test samples (for example-Among total growth, the highest growth rate was found in age groups >45 years in both Gram-positive and Gram-negative at 14 (26.9%) and 12 (23.1%)respectively.Least growth was found in an age group of 45 years....... etc) Tables:the table are well adjusted and most of them are changed in figure forms as in the final manuscripts and all the figures are of good quality.Point 4:all the antibiotics name through the manuscript have capital on the first letter.Point 1-5:the indicated points are all well captured on the final copy of our work.Please address all reviewers comments below paying careful attention to discrepancies in data in the manuscript, and methodologies that need to be clarified.Both reviewers have made good suggestions to improve the data presentation in the manuscript.Please do merge the data from figures 2-4 into one figure as suggested by reviewer 2. I appreciate that reproducing the plate images in the supplementary figures may not be possible if you no longer have the bacterial strains, but if it is possible better photographs would be appreciated, especially for the zone of clearance plates.Additionally, both reviewers highlight issues with structuring and legibility of the text that need to be thoroughly addressed during revision.
Version 2 REPORT TO REVIEWER.POINT 1:ANOVA Dunnett's multiple comparisons test (*P=0.05;**P=0.01;***P=0.001;****P=0.0001) was used throughout and this is indicate under sub title Data analysis in line 176-177.POINT 2:isolate 12583, 14421and 11985ofP.aeruginosa spp.Strains are the specific inoculum strain used to seed the microtiter plates in the biofilm assays as in line 151-152 POINT 3:Yes only four isolates are MDR but unfortunately one isolate was able to shows resistance to more than one antimicrobial classes and this is now well highlighted on this paper on line 226-227.POINT 4: